Axonal injury is a central component in many neurological disorders including trauma, stroke, and multiple sclerosis as well as neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's disease (PD). The overall goal of this research is to develop compartmentalized culture chambers using microfabrication techniques that isolate axons in culture to facilitate the study of axon growth, function and pathology. Specifically, the design and fabrication of microdevices will allow the quantitative delivery of pharmacological agents selectively to axons and allow their effects on axonal transport and growth to be examined in a controlled environment. By understanding the effects of toxins or drugs on axon growth and transport, we will gain mechanistic insight into the pathophysiology of neurodegenerative disorders and injury, which could in turn guide the development of therapeutics for treatment of degeneration or injury. The focus of this project is to develop open chamber microdevices that allow axonal isolation and the targeted application of drugs/toxins. Because patient data, genetic models, and in vivo and in vitro toxin studies all support a compelling role for axonal dysfunction in PD, we will use a well characterized PD model to study toxin effects in axons at risk in this disorder. Specifically, cultures of midbrain neurons from mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase (TH) promoter in our microdevices will allow us to test in real time the hypothesis that the Parkinson mimetic 6-OHDA (6-Hydroxydopamine) triggers changes in axonal transport resulting in the loss of axonal function and ultimately cell death. The specific aims of this proposal are to: (1) determine the design constraints for an open chamber design that allows easy cell seeding and axonal assays, which can be readily scaled for medium/high throughput cultures, (2) determine the design constraints for a novel microdevice that allows the application of a drug/toxin to axons (but not cell bodies) that are synapsing onto another population of neurons, and (3) use the new microdevices to test for the first time the effects of PD-mimetic 6-OHDA on mitochondrial trafficking in dopaminergic (DA) axons. PUBLIC HEALTH RELEVANCE: If the aims of this proposal are achieved, novel microdevices will become available to a large numbers of researchers trying to understand the role that axons play in neurological disease and injury. Establishing a causal role for axon dysfunction in neurodegenerative disorders such as Parkinson's and Alzheimer's disease is a challenging task given current methodological restraints. The tools proposed here will overcome current limitations in this field and further advance our knowledge in this area by providing unique platforms to study pharmacological and molecular mechanisms of disease and to screen for novel therapeutics.